福建养殖仿刺参抗氧化多肽的酶解工艺优化及其对过氧化氢诱导的血管内皮细胞EA.hy926损伤的保护作用

Optimization of Enzymatic Hydrolysis of Aquacultured Sea Cucumber Apostichopus japonicus in Fujian and Protective Effects of Enzymatic Hydrolysate against Hydrogen Peroxide in Human Vascular Endothelial Cells EA.hy926

目的:优化仿刺参抗氧化多肽的酶解工艺,并研究其对过氧化氢(hydrogen peroxide,H_2O_2)诱导的人脐静脉内皮细胞株EA.hy926损伤的保护效应。方法:以酶解产物的水解度、体外DPPH自由基清除率为指标,筛选出最适蛋白酶;在单因素实验基础上,选取温度、加酶量、pH作为影响因子,以体外DPPH自由基清除率为响应值,结合响应面试验优化酶解工艺条件;进一步探讨酶解多肽体外抗氧化活性。以MTS法检测低、中、高剂量组的仿刺参抗氧化多肽对H_2O_2诱导的血管内皮细胞损伤的保护作用,以MDA含量、SOD活力测定细胞氧化及抗氧化水平。结果:动物蛋白水解酶为最适蛋白酶,酶解工艺优化条件为:料液比1∶20 g/m L、酶解时间2 h、酶解温度50℃、加酶量7000 U/g、pH7.5,该条件下制备的仿刺参多肽体外DPPH自由基清除率为68.81%,与模型预测值(68.35%),相对误差为1%,回归模型可靠。酶解多肽对DPPH自由基、羟自由基(·OH)、超氧阴离子自由基(O_2^-·)和ABTS自由基(ABTS^+·)的半数抑制浓度IC_(50)分别是9.01、0.63、10.89和20.53 mg/m L,说明其具有较好的体外抗氧化活性。选取200μmol/L浓度H_2O_2建立细胞损伤模型,与H_2O_2组相比,中、高剂量组仿刺参抗氧化多肽能明显抑制H_2O_2诱导的血管内皮细胞氧化损伤,降低MDA含量,提高SOD活力。结论:采用动物蛋白水解酶酶解优化工艺制备的仿刺参抗氧化多肽对人脐静脉内皮细胞EA.hy926具有显著的保护作用并呈显著的剂量-效应关系。

Objective :To determine the optimal enzymatic hydrolysis conditions of polypeptides from Apostichopus japonicas and explore the protective effect of enzymatic hydrolysate against hydrogen peroxide (H2O2)induced oxidative stress in human vascular endothelial cell line(EA.hy926).Methods:The single factor methodology was carried out to determine the optimal temperature,enzyme volume and pH.The response surface methodology was used to optimize the enzymatic hydrolysisconditions.The in vitro antioxidant activities of enzymolysis polypeptide were investigated.The MTS method was performed to evaluated the protective effects of enzymatic hydrolysate against H2O2induced oxidative stress in EA.hy926.The MDA and SOD values were monitored.Results :Animal protease was the most suitable protease.The optimum conditions of enzymatic hydrolysis were as follows:Solid-liquid ratio was 1:20g/mL,the enzymatic hydrolysis time was 2h,the enzymatic hydrolysis temperature was 50℃,the enzyme concentration was 7000U/g,and the pH was 7.5.Under these conditions,the DPPH free radical scavenging rate of the peptides was 68.81%,and the relative error was 1% with the predicted value of the model(68.35%), which indicated the regression model was reliable.The IC50 inhibitory concentrations of DPPH radical,hydroxyl radical (·OH),superoxide anion radical(O2^·)and ABTS ^+· were 9.01,0.63,10.89 and 20.53mg/mL respectively,which showed that the enzymolysis polypeptide had good antioxidant activity in vitro.A cell injury model was established with a concentration of 200μmol/L H2O2.Compared with H2O2 group,the antioxidant peptides of middle and high dose groups could significantly inhibit oxidative damage of vascular endothelial cells induced by H2O2,reduce MDA content and increase SOD activity. Conclusion:The antioxidant peptides prepared by enzymatic hydrolysis of animal proteolytic enzymes had significant protective effect on human umbilical vein endothelial cells EA.hy926 and had a significant dose-effect relationship.